Haemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII) activity. The clinical manifestation is not on primary haemostasis as formation of the initial blood clot occurs normally. Rather the clot is unstable due to a lack of secondary thrombin formation and fibrin stabilization of the primary clot. The disease is treated by intravenous injection of FVIII which is either isolated from blood or produced recombinantly. Development of neutralizing antibodies (inhibitors) against FVIII occurs in approximately 20-40% of severe harmophilia A patients after FVIII administration, rendering further treatment with FVIII ineffective. Induction of inhibitors thus provides a major complication in haemophilia care.
Current treatment recommendations are moving from traditional on-demand treatment towards prophylaxis. The circulatory half life of endogenous FVIII bound to von Willebrand Factor (vWF) is 12-14 hours and prophylactic treatment is thus to be performed several times a week in order to obtain a virtually symptom-free life for the patients. Intravenous administration is for many, especially children and young persons, associated with significant inconvenience and/or pain.
Various methods have been employed in the development of a FVIII variant with significantly prolonged circulatory half life. A number of these methods relate to conjugation of FVIII with hydrophilic polymers such as e.g. PEG (poly ethylene glycol). WO03031464 discloses an enzymatic approach where PEG groups can be attached to glycans present on the polypeptide.
It has also been suggested to modulate low density lipoprotein receptor related protein (LRP) mediated clearance of FVIII in order to obtain a FVIII variant with a decreased rate of cellular uptake/clearance and thus an increased in vivo circulatory half life, but this approach has been hampered by the apparent massive redundancy of potential LRP binding sites present on the surface of FVIII and uncertainty on the role of these. Furthermore, some of these sites are in close vicinity to regions critical for FVIII:C activity, and a lowered LRP binding may be accompanied by a substantial loss of activity which makes the FVIII variant less attractive as a therapeutic agent. Interaction of LRP and related receptors with its ligands is thought to involve lysine residues on the surface of the ligand docking in an acidic “necklace” in the receptor (Mol Cell 2006; 22: 277-283). It has furthermore been suggested that hydrophobic residues, in combination with lysine residues, may be involved in interaction with LRP-family members (FEBS J 2006; 273: 5143-5159, J Mol Biol 2006; 362: 700-716) and it could therefore be speculated if modification of these hydrophobic residues, in addition to critical lysine residues or other positively charged residues, could result in decreased interaction with LRP family members and potentially prolonged and/or decreased clearance.
In order to be of therapeutic interest, FVIII variants should retain FVIII procoagulant function. It thus follows that there is a need in the art for specific FVIII variants with maintained FVIII activity and a significantly prolonged in vivo circulatory half life and/or reduced immunogenicity.